CN101857872A - Method for Replacing Antigen Determinant Region of Influenza A Virus - Google Patents
Method for Replacing Antigen Determinant Region of Influenza A Virus Download PDFInfo
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Abstract
本发明公开一种甲型流感病毒抗原决定区更换方法。本发明针对抗原决定区的位置，利用和设计酶切位点，能够实现同一亚型或不同亚型流感病毒株相互之间抗原决定区序列的整体或部分更换，并利用8质粒反向基因操作系统拯救出抗原性经过人为定向改变的重组流感病毒，在流感病毒减毒性活疫苗的快速制备和病毒载体在基因治疗中多次重复使用方面具有重大的应用价值。The invention discloses a method for replacing the antigenic determinant region of influenza A virus. The present invention aims at the position of the antigen-determining region, utilizes and designs the enzyme cutting site, can realize the whole or partial replacement of the sequence of the antigen-determining region between the same subtype or different subtype influenza virus strains, and utilizes 8 plasmid reverse gene manipulation The system rescues the recombinant influenza virus whose antigenicity has been artificially changed, which has great application value in the rapid preparation of attenuated live vaccine of influenza virus and the repeated use of virus vector in gene therapy.
Description
Technical field
The invention belongs to bioengineering field, relate in particular between the different strains of a kind of influenza A virus the replacement method of influenza A Virus antigenic determinant of antigenicity fragment mutual alternative on the HA gene.
Background technology
Influenza is called for short influenza, is familiar with the history in year surplus in the of existing 100 by people, and every autumn and winter and winter-spring season festival-gathering just take place popular, becomes one of the most serious transmissible disease in the whole world.It is global several times popular on a large scale that 20th century took place, and causes a large amount of human mortalities, brings very grave disaster to human society.For example, because of the dead number of spanish influenzas in 1918 (H1N1) is estimated to surpass 5,000 ten thousand, the Mao flu (H3N2) of the Asia influenza of nineteen fifty-seven (H2N2) and nineteen sixty-eight also has millions of people's death, and the death toll that this disease causes is considerably beyond because of the dead number of war.2009, be originated in Mexican new Influenza A H1N1 and wreak havoc in the whole world.
Influenza virus is the pathogenic agent that causes influenza, belongs to the segmented RNA viruses of the negative justice of orthomyxovirus section sub-thread, and different according to nucleoprotein and M protein antigenicity are divided into first, second, the third three types.Wherein the Influenza B virus antigenicity is relatively stable, and the vaccine prevention effect is remarkable, and the self-contained antibody of general population has had immanoprotection action preferably.The influenza virus C antigenicity is stable, mostly is Sporadic cases, does not cause popularly, endangers little.But characteristics such as influenza A virus has very easily variation, hypotype is numerous, infectivity is strong, host range is wide are one of current the most unmanageable zymads.Cause world wide pandemic all be influenza A virus, the avian influenza virus of hypotypes such as human infection H5N1, H9N2, H7N7 also belongs to the influenza A virus category over past ten years.Influenza A virus is different with the antigenicity of neuraminidase (NA) by its surface glycoprotein hemagglutinin (HA), can be divided into 16 HA hypotypes and 9 NA hypotypes again, and on HA and NA, exist a plurality of antigen determining area, HA proteantigen determining area as the H1 hypotype is divided into Sa, Sb, Ca1, Ca2 and Cb, and the HA proteantigen determining area of H3 subtype virus is divided into A, B, C, D and E.The influenza A virus antigenic variation is divided into antigenic drift and antigenic shift two classes.Antigenic drift refers to origination point or local variation gradually, causes that the middle and small scale of influenza is popular, it is generally acknowledged that antigenic drift makes influenza virus occur a new variant in per 1～3 year; Antigenic shift refers to big antigenic variation, and the variation result can produce new different subtype, and its change is closely related with the virulence of virus.Because the crowd lacks corresponding antibodies to new hypotype, usually can cause big popular.Therefore; research and development have brought great difficulty to characteristics such as influenza A virus that hypotype is numerous and antigenicity thereof very easily make a variation to vaccine; conventional inactivated vaccine can not cover the strains of influenza viruses of variation at any time fully, and often can the escape provide protection and causing of vaccine of virus is very popular.The currently available vaccines, existing vaccines preventative strategies can presents a kind of tangible hysteretic state, and in the face of new strain or new hypotype, original vaccine can't provide enough effectively protection, need research and develop the specific aim vaccine at short notice.Along with the develop rapidly of Protocols in Molecular Biology, the birth of cdna reverse operating system is with constantly perfect, make people's directional transformation influenza virus, utilize influenza virus to become possibility for mankind service.The present invention proposes the platform that a kind of influenza antigen is changed, by cdna reverse operating system the influenza virus antigenicity is carried out the people for a change, the influenza vaccines of realizing exploitation strain rate effect ability, and be to have great application value in the gene therapy of carrier with the influenza virus.
The reverse genetic technology has vital role in the virological investigation field, and one of them is exactly the rescue and the transformation of virus.Utilize the reverse genetic technology, people can transform virus as required, artificially save new virus, wait as virus vector or vaccine strain and use.U.S. scientist in 1989 at first utilizes cdna reverse operating system and successfully obtains influenza virus through artificial recombination.Neumann in 1999 etc. utilize cdna reverse operating system obtaining important breakthrough aspect the rescue influenza virus, and the full pUC pUC of employing has been broken away from the use of helper virus.Along with development of technology, plasmid quantity is by the most at first 17 constantly reductions.At present the most frequently used is 8 pUC pUCs, i.e. the double-promoter carrier system of rna plymerase i/II (Pol I/II).Cdna reverse operating system more and more is widely used, and comprises the molecular biology research of influenza virus, the development of influenza vaccines and the trial of influenza vectors etc., makes the novel influenza vaccines of research and development become possibility.According to the influenza virus biological characteristics, can carry exogenous genetic fragment at its HA, NA and NS gene, influenza virus is also more and more paid close attention in field of gene as genophore.
Gene therapy is meant people's normal gene or medicative gene is imported defective or the performance therapeutic action of people's somatic target cell to correct gene by certain way, thereby reaches the biomedical new technology of treatment disease purpose.Gene therapy mainly is to treat those human health is threatened serious disease at present, comprising: inherited disease, malignant tumour, cardiovascular disorder, infectious diseases etc.Yet the carrier of selecting to be fit to then is the key factor that treatment is achieved success.The gene therapy common carrier can be divided into non-virus carrier and virus vector two big classes, non-virus carrier transfection ability wherein, and research of animal level and clinical application all are subjected to huge restriction; And virus vector has advantages such as efficiency of infection height, host range are wide, stability is strong and attracts people's attention.In recent years; Chinese scholars utilizes retroviral vector, adenovirus carrier, lentiviral vectors, vaccinia virus vector etc. to obtain bigger progress in gene therapy; but the immanoprotection action because of body after being to use virus vector to treat first makes that reusable curative effect is undesirable; this science difficult problem is restricting virus vector promoting the use of on clinical treatment all the time; also there is this problem in the most sophisticated at present adenovirus carrier, still can not effectively solve.Though and the numerous influenza virus of hypotype has brought great difficulty for the vaccine research and development, the repeated use for virus vector in the gene therapy process provides favourable condition.Influenza vectors is escaped immune tracking by antigenic replacing when reusing, reusing undesirable this difficult problem of effect thereby be expected to effectively to solve virus vector.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of replacement method of influenza A Virus antigenic determinant is provided, the present invention realized between the different subtype virus and the different strains of same hypotype between whole replace or part is replaced of antigenic region, the application in gene therapy provides effective antigenicity switch technology platform for influenza attenuation living vaccine and influenza vectors.The transcription vector of related loading influenza antigen region sequence is pPolI among the present invention, and total length HA gene is inserted between two BsmBI restriction enzymes of pPolI enzyme recognition site.The HA gene fragment of inserting is by sequential analysis and Nucleotide rite-directed mutagenesis, set some restriction enzyme sites (these restriction enzyme sites are not present on the carrier framework), on aminoacid sequence, antigenic region is divided into several parts, be applicable to the replacing of antigen determining area sequence.
The objective of the invention is to be achieved through the following technical solutions:
A kind of replacement method of influenza A Virus antigenic determinant, this method may further comprise the steps:
(1) clone obtains influenza virus total length hemagglutinin gene from virus-culturing fluid, these hemagglutinin gene two ends comprise complete non-translational region sequence, and be inserted among the transcription vector pPolI, construct the reorganization transcription vector that contains hemagglutinin gene, as one of recombinant plasmid of cdna reverse operating system.
(2) on the HA gene order, select appropriate site to carry out rite-directed mutagenesis, make up the restriction enzyme site that makes new advances, be applicable to the replacing of antigen determining area.
(3) the mutual replacing of HA gene antigen determining area sequence between the different antigenicity influenza viruses: described antigen determining area sequence is changed mutually and is comprised two contents, and one is the integral replacing of antigen determining area sequence, or the replacing of whole piece HA gene; One is the part replacing of antigen determining area sequence.Replacing can be carried out between the corresponding sequence of the different strains of same hypotype, also can carry out between the corresponding sequence of the different strains of different subtype.
(4) HA gene antigen determining area sequence is changed the rescue of back recombinant virus: utilize the above-mentioned recombinant plasmid that HA gene antigen district dna sequence dna is changed that carried out, cdna reverse operating system by 8 plasmids, cotransfection is to the 293T cell, and with mdck cell propagation, save out the recombinant influenza of antigenic shift.
The invention has the beneficial effects as follows,
1. the quick structure of attenuation live influenza virus vaccine candidate strain
On the one hand, by above-mentioned antigenic shift platform, can be the antigen of low pathogenicity strain with the antigenic shift of highly pathogenic strain, virus strain is carried out attenuation; On the other hand, by above-mentioned antigenic shift platform, can on purpose change the antigenicity of strains of influenza viruses, influenza epidemic situation in the face of burst, the antigen determining area of pathogenic agent is changed on original temperature sensitivity attenuated live vaccine, make recombinant virus keep the low toxicity feature on the one hand, have the antigenicity similar to pathogenic agent on the one hand, the attenuated live vaccine that production is had the strain rate effect ability is significant.
2. solving virus vector can not reusable bottleneck problem
Gene therapy will be captured in future has boundless application prospect aspect the multiple difficult and complicated illness such as malignant tumour, inherited disease.The genome of self can be carried in the cell during virus infection human body cell, and utilize the interior material of human body cell to finish self-reproduction, finally cause human diseases.Utilize this characteristic, remove in the viral genome and pathogenic relevant gene, keep it and carry the function that genome enters human body cell, assemble the foreign gene of coideal again, promptly become a kind of virus vector.Virus vector has advantages such as efficiency of infection height, host range is wide, stability is strong and attracts people's attention, and has unique advantage in gene therapy.In recent years; Chinese scholars utilizes retroviral vector, adenovirus carrier, lentiviral vectors, vaccinia virus vector etc. to obtain bigger progress in gene therapy; but the immanoprotection action because of body after being to use virus vector to treat first makes that reusable curative effect is undesirable, and this science difficult problem is restricting virus vector promoting the use of on clinical treatment all the time.Though the most sophisticated adenovirus carrier of research has been carried out many work clinically at present, can't effectively break through repeatedly not good this predicament of result of use on same individuality all the time.And the antigen of influenza virus is various, and weak these characteristics of immunological cross-reaction are brought hope for solving this difficult problem between a plurality of hypotypes.Can carry out autotelic antigenic shift to influenza virus by the present invention, construct the various influenza vectors system of a series of antigens, when reusing, utilize different antigenic influenza vectors repeatedly to treat, escape immune tracking, reusing undesirable this difficult problem of effect thereby can effectively solve virus vector.
Description of drawings
Fig. 1 is that the PCR of recombinant plasmid pPolI/WSN-HA/Xba82/H1 identifies figure, and among the figure, arrangement from left to right is respectively: M.DL2000; 1. the PCR product of total length HA gene; 2.XbaI and the PCR product of dna fragmentation between the SacI restriction enzyme site; 3.SacI and the PCR product of dna fragmentation between the NcoI restriction enzyme site; 4.NcoI and the PCR product of dna fragmentation between the EcoRI restriction enzyme site; M.DL2000.
Fig. 2 cuts evaluation figure for the enzyme of recombinant plasmid pPolI/WSN-HA/Xba82/H1, and among the figure, arrangement from left to right is respectively: M.DL2000; 1.pPolI/WSN-HA/Xba82/H1; 2.pPolI/WSN-HA/Xba82/H1 the NheI enzyme cut; 3.pPolI/WSN-HA/Xba82/H1 XbaI and SacI double digestion; 4.pPolI/WSN-HA/Xba82/H1 SacI and NcoI double digestion; 5.pPolI/WSN-HA/Xba82/H1 NcoI and EcoRI double digestion; M.DL2000.
Fig. 3 is that the PCR of recombinant plasmid pPolI/Memphis-HA/Pst35/H3 identifies figure, among the figure, arranges from left to right and is respectively: DNA Ladder M.200bp; M.1kb DNA Ladder; 1. the PCR product of total length HA gene; 2.PstI and the PCR product of dna fragmentation between the HindIII restriction enzyme site; 3.HindIII and the PCR product of dna fragmentation between the XbaI enzyme cutting site; 4.XbaI and the PCR product of dna fragmentation between the NsiI restriction enzyme site; 5.NsiI and the PCR product of dna fragmentation between the SphI restriction enzyme site; 6.SphI and the PCR product of dna fragmentation between the XhoI restriction enzyme site.
Fig. 4 cuts evaluation figure for the enzyme of recombinant plasmid pPolI/Memphis-HA/Pst35/H3, and among the figure, arrangement from left to right is respectively: 1.pPolI/Memphis-HA/Pst35/H3; 2.pPolI/Memphis-HA/Pst35/H3 the NheI enzyme cut; 3.pPolI/Memphis-HA/Pst35/H3 PstI and HindIII double digestion; 4.pPolI/Memphis-HA/Pst35/H3 HindIII and XbaI double digestion; 5.pPolI/Memphis-HA/Pst35/H3 XbaI and NsiI double digestion; 6.pPolI/Memphis-HA/Pst35/H3 NsiI and SphI is two cuts; 7.pPolI/Memphis-HA/Pst35/H3 SphI and XhoI double digestion; M.200bp DNA Ladder; M.1kb DNA Ladder.
Fig. 5 is that the PCR of recombinant plasmid pPolI/HK312-HA/Xho68/H5 identifies figure, among the figure, arranges from left to right and is respectively: DNA Ladder M.200bp; 1. the PCR product of total length HA gene; 2.XhoI and the PCR product of dna fragmentation between the SacI restriction enzyme site; 3.SacI and the PCR product of dna fragmentation between the BamHI restriction enzyme site; 4.BamHI and the PCR product of dna fragmentation between the SphI restriction enzyme site; 5.SphI and the PCR product of dna fragmentation between the AflII restriction enzyme site; M.1kb DNA Ladder.
Fig. 6 cuts evaluation figure for the enzyme of recombinant plasmid pPolI/HK312-HA/Xho68/H5, and among the figure, arrangement from left to right is respectively: the NheI enzyme of 1.pPolI/HK312-HA/Xho68/H52.pPolI/HK312-HA/Xho68/H5 is cut; 3.pPolI/HK312-HA/Xho68/H5 XhoI and SacI double digestion; 4.pPolI/HK312-HA/Xho68/H5 SacI and BamHI double digestion; 5.pPolI/HK312-HA/Xho68/H5 BamHI and SphI double digestion; 6.pPolI/HK312-HA/Xho68/H5 SphI and AflII double digestion; M.200bp DNA Ladder; M.1kb DNA Ladder.
Embodiment
Summary of the invention
Replacement method of influenza A Virus antigenic determinant of the present invention may further comprise the steps:
1. the clone obtains influenza virus total length hemagglutinin gene from virus-culturing fluid, the hemagglutinin gene two ends comprise complete non-translational region sequence, and be inserted among the transcription vector pHH21 (being called pPolI again), construct the reorganization transcription vector that contains hemagglutinin gene, as one of recombinant plasmid of cdna reverse operating system.
(1) H1 subtype influenza virus
With H1 subtype influenza virus strain A/WSN/1933 (H1N1) is material, the RT-PCR method obtains the total length HA gene fragment that complete non-translational region is contained at two ends, be cloned between two restriction enzyme site BsmBI of described transcription vector pPolI this recombinant plasmid called after pPolI/WSN-HA/H1.
(2) H3 subtype influenza virus
With H3 subtype influenza virus strain A/Memphis/1/1971 (H3N2) is material, the RT-PCR method obtains the total length HA gene fragment that complete non-translational region is contained at two ends, be cloned between two restriction enzyme site BsmBI of described transcription vector pPolI this recombinant plasmid called after pPolI/Memphis-HA/H3.
(3) H5 subtype influenza virus
With H5 subtype influenza virus strain A/duck/HK/312/78 (H5N3) is material, the RT-PCR method obtains the total length HA gene fragment that complete non-translational region is contained at two ends, be cloned between two restriction enzyme site BsmBI of described transcription vector pPolI this recombinant plasmid called after pPolI/HK312-HA/H5.
2. on the HA gene order, select appropriate site to carry out rite-directed mutagenesis, make up the restriction enzyme site that makes new advances, be applicable to the replacing of antigen determining area.
(1) .H1 subtype influenza virus
HA gene order to above-mentioned recombinant plasmid pPolI/WSN-HA/H1 is carried out the Nucleotide rite-directed mutagenesis, utilize original part restriction enzyme site simultaneously, construct the recombinant plasmid that XbaI is arranged, SacI is arranged on 124, has on 267 NcoI on 82 of HA gene, the EcoRI restriction enzyme site is arranged on 412, called after pPolI/WSN-HA/Xba82/H1 is applicable to the replacing of antigen determining area.Between the XbaI of above-mentioned recombinant plasmid pPolI/WSN-HA/Xba82/H1 and the SacI restriction enzyme site at a distance of 126 Nucleotide; Between SacI and the NcoI restriction enzyme site at a distance of 429 Nucleotide; Between NcoI and the EcoRI restriction enzyme site at a distance of 435 Nucleotide (Fig. 1, Fig. 2).
(2) .H3 subtype influenza virus
HA gene order to above-mentioned recombinant plasmid pPolI/Memphis-HA/H3 is carried out the Nucleotide rite-directed mutagenesis, utilize original part restriction enzyme site simultaneously, construct the recombinant plasmid that PstI is arranged, HindIII is arranged on 109, has on 235 XbaI on 35 of HA gene, NsiI is arranged on 298, has on 321 SphI, the XhoI restriction enzyme site is arranged on 425, called after pPolI/Memphis-HA/Pst35/H3 is applicable to the replacing of antigen determining area.Between the PstI of above-mentioned recombinant plasmid pPolI/Memphis-HA/Pst35/H3 and the HindIII restriction enzyme site at a distance of 293 Nucleotide; Between HindIII and the XbaI enzyme cutting site at a distance of 378 Nucleotide; Between XbaI and the NsiI restriction enzyme site at a distance of 267 Nucleotide; Between NsiI and the SphI restriction enzyme site at a distance of 69 Nucleotide; Between SphI and the XhoI restriction enzyme site at a distance of 312 Nucleotide (Fig. 3, Fig. 4).
(3) H5 subtype influenza virus
HA gene order to above-mentioned recombinant plasmid pPolI/HK312-HA/H5 is carried out the Nucleotide rite-directed mutagenesis, utilize original part restriction enzyme site simultaneously, construct the recombinant plasmid that XhoI is arranged, SacI is arranged on 149, has on 193 BamHI on 68 of HA gene, SphI is arranged on 306, restriction enzyme sites such as AflII are arranged on 468, called after pPolI/HK312-HA/Xho68/H5 is applicable to the replacing of antigen determining area.Between the XhoI of above-mentioned recombinant plasmid pPolI/HK312-HA/Xho68/H5 and the SacI restriction enzyme site at a distance of 243 Nucleotide; Between SacI and the BamHI restriction enzyme site at a distance of 132 Nucleotide; Between BamHI and the SphI restriction enzyme site at a distance of 339 Nucleotide; Between SphI and the AflII restriction enzyme site at a distance of 486 Nucleotide (Fig. 5, Fig. 6).
3. the mutual replacing of HA gene antigen determining area sequence between the different antigenicity influenza viruses
Described antigen determining area sequence is changed mutually and is comprised two contents, and one is the integral replacing of antigen determining area sequence, or the replacing of whole piece HA gene; One is the part replacing of antigen determining area sequence.Replacing can be carried out between the corresponding sequence of the different strains of same hypotype, also can carry out between the corresponding sequence of the different strains of different subtype.
Being substituted into low pathogenicity influenza virus A/duck/HK/312/78 (H5N3) with the antigen fragment of highly pathogenic virus strain A/Vietnam/1203/04 (H5N1) below is example.
(1). the integral replacing of influenza virus HA gene antigen determining area sequence
When carrying out the replacing of whole piece gene, with HA gene fragment excision among the above-mentioned recombinant plasmid pPolI/HK312-HA/Xho68/H5, insert the corresponding sequence of the HA gene fragment of A/Vietnam/1203/04 (H5N1) strain, the HA gene that comprises in the feasible new recombinant plasmid that makes up derives from A/Vietnam/1203/04 (H5N1) strain fully, plays the effect of changing fully.
(2). the part of influenza virus HA gene antigen determining area sequence is changed
When carrying out antigenic region when partly changing, then in XhoI, the SacI of above-mentioned recombinant plasmid pPolI/HK312-HA/Xho68/H5, BamHI, SphI, AflII restriction enzyme site, select two suitable restriction enzyme sites according to the antigenic region size and location of required replacement.As the antigenic region of required replacing between restriction enzyme site SacI and SphI, then can adopt pcr amplification or enzyme blanking method to obtain the dna fragmentation of A/Vietnam/1203/04 (H5N1) strain HA gene respective section, be inserted among the above-mentioned recombinant plasmid pPolI/HK312-HA/Xho68/H5, substitute original corresponding gene order, make new recombinant plasmid HA gene SacI that makes up and the dna sequence dna between the SphI restriction enzyme site derive from strain A/Vietnam/1203/04 (H5N1), play the effect that part is changed.
Adopt above-mentioned integral replacing and part replacing method, carry out the mutual alternative of antigen determining area between can strains of influenza viruses any two same hypotypes or different subtype.
4.HA gene antigen determining area sequence is changed the rescue of back recombinant virus
Utilize the above-mentioned recombinant plasmid that HA gene antigen district dna sequence dna is changed that carried out, by the cdna reverse operating system of 8 plasmids, cotransfection is to the 293T cell, and breeds with mdck cell, saves out the recombinant influenza of antigenic shift.
The invention will be further described below in conjunction with specific embodiment.But these embodiment must not be interpreted as and go up limitation of the present invention in all senses.
The clone of hemagglutinin gene
In Biohazard Safety Equipment, extract viral RNA by the method that Initrogen company specification sheets provides with Trizol, with V13 reverse transcriptase primer, by the synthetic viral cDNA of ThermoScript II MMLV.And be template with above-mentioned viral cDNA, obtain the total length HA gene fragment that complete non-translational region is contained at two ends by V12 and two primer PCRs amplifications of V13.Be template with this total length HA gene fragment again, obtain the HA full-length gene, introduce a BsmBI restriction enzyme site separately at the two ends of gene simultaneously by 5BmHA and two primer amplifications of 3BmHA.Concrete be exactly: the primer of H1 subtype influenza virus (A/WSN/1933) the total length HA gene that is used to increase is V12 and V13, be template again with the amplified production, respectively introduce a BsmBI restriction enzyme site by primer 5BmHA and 3BmHA at HA gene two ends, be used for being cloned into transcription vector pPolI; The primer of H3 subtype influenza virus (A/Memphis/1/1971) total length HA gene of being used to increase is V12 and V13, be template again with the amplified production, respectively introduce a BsmBI restriction enzyme site by primer 5BmHA and 3BmHA at HA gene two ends, be used for being cloned into transcription vector pPolI; The primer of H5 subtype influenza virus (A/duck/HK/312/78) total length HA gene of being used to increase is V12 and V13, be template again with the amplified production, respectively introduce a BsmBI restriction enzyme site by primer 5BmHA and 3BmHA at HA gene two ends, be used for being cloned into transcription vector pPolI.
The primer sequence of above-mentioned PCR is respectively:
V12：AGCAAAAGCAGG；
V13：AGTAGAAACAAGG；
5BmHA：TATTCGTCTCAGGGAGCAAAAGCAGGGG
3BmHA：ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT。
Total length HA gene clone is to transcription vector pPolI
The total length HA gene that embodiment 1 described two ends is all had the BsmBI restriction enzyme site is cut with the BsmBI enzyme, and the transcription vector pPolI that enzyme is cut behind the product purification with the BsmBI enzyme is cut is connected, and screening obtains behind the recombinant plasmid and carries out determined dna sequence.In pPolI, insert the recombinant plasmid called after pPolI/WSN-HA/H1 of H1 hypotype A/WSN/1933 (H1N1) strain HA gene; In pPolI, insert the recombinant plasmid called after pPolI/Memphis-HA/H3 of H3 hypotype A/Memphis/1/1971 (H3N2) strain HA gene; In pPolI, insert the recombinant plasmid called after pPolI/HK312-HA/H5 of H5 hypotype A/duck/HK/312/78 (H5N3) strain HA gene.
Rite-directed mutagenesis is introduced new restriction enzyme site
By the method for rite-directed mutagenesis, in embodiment 2 described recombinant plasmids, introduce new restriction enzyme site, be used for antigenic shift.To introduce the XbaI restriction enzyme site in described recombinant plasmid pPolI/WSN/H1 is example, and concrete steps are as follows:
With pPolI/WSN/H1 is template, is the point mutation primer with H1XbaHA82F and H1XbaHA82R, carries out pcr amplification.Amplification condition is 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of extension 6min, totally 18 circulations, last 72 ℃ are extended 10min.Digest 1h with restriction enzyme DpnI at 37 ℃ behind the amplified production purifying, ethanol/sodium-acetate post precipitation is dissolved in the 20 μ l distilled waters, gets 1 μ l transformed into escherichia coli DH5 α, and the screening mutant clon also carries out sequencing, confirms to introduce the XbaI enzyme cutting site.HA gene through point mutation contains XbaI, SacI, NcoI, EcoRI restriction enzyme site (Fig. 2).
With H3Pst35HAF and H3Pst35HAR is the point mutation primer, use aforesaid method and carry out rite-directed mutagenesis, introduce the PstI restriction enzyme site for 35 at described recombinant plasmid pPolI/Memphis-HA/H3 hemagglutinin gene, the HA gene of process rite-directed mutagenesis contains restriction enzyme site PstI, HindIII, XbaI, NsiI, SphI, XhoI.
With H5XhoHA68F and H5XhoHA68R is the point mutation primer, use aforesaid method and carry out rite-directed mutagenesis, introduce Xho I restriction enzyme site for 68 at described recombinant plasmid pPolI/HK312-HA/H5 hemagglutinin gene, the HA gene of process rite-directed mutagenesis contains restriction enzyme site XhoI, SacI, BamHI, SphI, AflII.
The primer of above-mentioned rite-directed mutagenesis is respectively:
H1XbaHA82F：GGGAAATCTAGAATGCGAC；
H1XbaHA82R：GTCGCATTCTAGATTTCCC；
H3Pst35HAF：CATCCTGCAGTGCCAAAC；
H3Pst35HAR：GTTTGGCACTGCAGGATG；
H5XhoHA68F：CCTCTCATCTCGAGGGATTG；
H5XhoHA68R：CAATCCCTCGAGATGAGAGG
Antigenic shift---full gene is changed
With the HA gene integral replacing of highly pathogenic virus strain A/Vietnam/1203/04 (H5N1) in low pathogenicity strain A/duck/HK/312/78 (H5N3).For strains of influenza viruses A/Vietnam/1203/04 (H5N1), according to embodiment 1 described hemagglutinin gene cloning process, increase to such an extent that each have the complete HA gene (non-translational region that comprises 5 ' end and 3 ' end) of a BsmBI restriction enzyme site to two ends, the BsmBI enzyme is cut afterwards and is carried out purifying.With BsmBI described recombinant plasmid pPolI/HK312-HA/H5 is carried out enzyme simultaneously and cut, remove after the HA gene fragment derive from A/duck/HK/312/78 (H5N3) and carry out purifying.Both connect back screening recombinant clone, and the HA gene of the recombinant plasmid of acquisition (called after pPolI/VN1203-HA/H5) derives from A/Vietnam/1203/04 (H5N1) fully, and the HA gene obtains integral replacing.
Antigenic shift---incomplete antigen district changes
The incomplete antigen fragment of highly pathogenic influenza virus A/Vietnam/1203/04 (H5N1) is substituted among low pathogenicity influenza virus A/duck/HK/312/78 (H5N3), with the example of being replaced by of antigenic region sequence between SacI and two restriction enzyme sites of SphI.
By sequential analysis, the corresponding position of A/Vietnam/1203/04 (H5N1) and two virus strain of A/duck/HK/312/78 (H5N3) all has SacI and two restriction enzyme sites of SphI.Obtain the dna fragmentation between SacI and two restriction enzyme sites of SphI in A/Vietnam/1203/04 (H5N1) the virus strain HA gene by pcr amplification or double digestion method, and carry out purifying.Use SacI and SphI double digestion recombinant plasmid pPolI/HK312-HA/Xho68/H5 simultaneously, carry out purifying behind the dna fragmentation between removal SacI and the SphI site.Above-mentioned two purified dna fragmentations are connected the screening recombinant clone.In the recombinant plasmid that obtains, the gene order on the HA gene between SacI and the SphI site derives from virus strain A/Vietnam/1203/04 (H5N1), and the antigenic region sequence of HA gene obtains part and changes.
Rescue obtains the recombinant virus that the HA gene antigen is changed
Influenza virus rescue is based on 8 plasmid cdna reverse operating systems, and cotransfection is to the 293T cell, and breeds with mdck cell.8 plasmids are respectively pPol-II/I-Ann60-PB2, pPol-II/I-Ann60-PB1, pPol-II/I-Ann60-PA, pPol-II/I-Ann60-NP, pPol-II/I-Ann60-HA, pPol-II/I-Ann60-NA, pPol-II/I-Ann60-M, pPol-II/I-Ann60-NS (referring to sick the 33rd the 5th phase of volume of learning the loimology magazine of international popular, " structure of modified version cdna reverse operating system and the seed selection of influenza virus variant " literary composition).
If will obtain the recombinant influenza of HA gene through antigenic shift, then adopt above-mentioned cdna reverse operating system, wherein plasmid pPol-II/I-Ann60-HA can carry out the selectivity replacement as required.For example the HA of recombinant virus to be replaced by fully the HA of strain A/Vietnam/1203/04 (H5N1), then as long as pPol-II/I-Ann60-HA is replaced to pPolI/VN1203-HA/H5.
Claims (3)
1. replacement method of influenza A Virus antigenic determinant, it is characterized in that: this method may further comprise the steps:
(1) clone obtains influenza virus total length hemagglutinin gene from virus-culturing fluid, these hemagglutinin gene two ends comprise complete non-translational region sequence, and be inserted among the transcription vector pHH21 (being called pPolI again), construct the reorganization transcription vector that contains hemagglutinin gene, as one of recombinant plasmid of cdna reverse operating system.
(2) on the HA gene order, select appropriate site to carry out rite-directed mutagenesis, make up the restriction enzyme site that makes new advances, be applicable to the replacing of antigen determining area.
(3) the mutual replacing of HA gene antigen determining area sequence between the different antigenicity influenza viruses.
(4) HA gene antigen determining area sequence is changed the rescue of back recombinant virus.
2. according to the described replacement method of influenza A Virus antigenic determinant of claim 1, it is characterized in that: in the described step (3), described antigen determining area sequence is changed mutually and is comprised two contents, and one is the integral replacing of antigen determining area sequence, or the replacing of whole piece HA gene; One is the part replacing of antigen determining area sequence.Replacing can be carried out between the corresponding sequence of the different strains of same hypotype, also can carry out between the corresponding sequence of the different strains of different subtype.
3. according to claim 1 or 3 described replacement method of influenza A Virus antigenic determinant, it is characterized in that: described step (4) is specially: utilize the above-mentioned recombinant plasmid that HA gene antigen district dna sequence dna is changed that carried out, cdna reverse operating system by 8 plasmids, cotransfection is to the 293T cell, and with mdck cell propagation, save out the recombinant influenza of antigenic shift.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102180927A (en) * | 2011-03-25 | 2011-09-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for obtaining influenza virus nucleic acid in influenza sample and special primer thereof |
| CN109073639A (en) * | 2016-02-03 | 2018-12-21 | Cg探索股份有限公司 | The composition of the influenza hemagglutinin of mature cleavage site with heterologous epitope and/or change |
| CN113801205A (en) * | 2021-09-13 | 2021-12-17 | 湖南大学 | A kind of transformation method for improving the antigenicity of influenza A virus H1 trimeric protein |
| WO2023236822A1 (en) * | 2022-06-08 | 2023-12-14 | 中科南京生命健康高等研究院 | Development and use of h5n6 avian influenza broad-spectrum vaccine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180927A (en) * | 2011-03-25 | 2011-09-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for obtaining influenza virus nucleic acid in influenza sample and special primer thereof |
| CN109073639A (en) * | 2016-02-03 | 2018-12-21 | Cg探索股份有限公司 | The composition of the influenza hemagglutinin of mature cleavage site with heterologous epitope and/or change |
| CN109073639B (en) * | 2016-02-03 | 2022-04-01 | Cg探索股份有限公司 | Compositions of influenza hemagglutinin with heterologous epitopes and/or altered mature cleavage sites |
| CN113801205A (en) * | 2021-09-13 | 2021-12-17 | 湖南大学 | A kind of transformation method for improving the antigenicity of influenza A virus H1 trimeric protein |
| CN113801205B (en) * | 2021-09-13 | 2024-02-23 | 湖南大学 | Transformation method for improving antigenicity of H1 trimer protein of influenza A virus |
| WO2023236822A1 (en) * | 2022-06-08 | 2023-12-14 | 中科南京生命健康高等研究院 | Development and use of h5n6 avian influenza broad-spectrum vaccine |
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